A PHYfOCHEMICAL INVESTIGATION OF MEMBERS OF THE HYACINTHACEAE FAMILY AND BIOLOGICAL SCREENING OF HOMOISOFLAVANONES AND STRUCTURALLY RELATED COMPOUNDS BY KAREN DU TOIT B.Pharm., MSc. (Pharmaceutical Chemistry) (Potchefstroom) Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Pure and Applied Chemistry, University of KwaZulu-Natal, Durban 2004 Sou (])eo 9{oria PREFACE The phytochemical investigation described in this thesis was carried out in the School of Pure and Applied Chemistry, University of KwaZulu-Natal, Durban and in the Chemistry Department, University of KwaZulu-Natal, Pietermaritzburg. The biological assays were carried out in the Research Centre for Plant Growth and Development, University of KwaZulu-Natal, Pietermaritzburg and the quantitative structure-activity relationship studies were performed in the Pharmaceutical Chemistry Department, Potchefstroom Campus, North-West University, South Africa. This study was supervised by Professor D.A. Mulholland and Professor S.E. Drewes. The study represents original work by the author and has not been submitted in any other form to another university. Where use was made of the work of others, it has been duly acknowledged in the text. Signed: K. du Toit B.Pharm., M.Sc. (Pharmaceutical Chemistry), (Potchefstroom) I hereby certify that the above statement is correct. Signed: .4.(~~.... Professor D.A. Mulholland Professor S.E. Drewes (Supervisor) (Co-supervisor) 2 Acknowledgements The input of many people made this study possible. I wish to sincerely thank the following persons: • Professor Dulcie Mulholland at the Chemistry Department of the KwaZulu-Natal University, Durban, who has been my supervisor. A friend of mine once said that professor Mulholland reminds him of a giraffe as she has the ability to look ahead and to plan well. I would like to add that she also reminds me of an ant and an elephant - she has the ability to work very hard and she does not forget a promise. Thank you, Dulcie, for putting a lot of hard work into this thesis and for helping me to finish it! Thank you also to Mr Dhillip Jagjivan for the NMR spectra and Mr Brett Parel for helping with the Mass Spectra and for providing my laboratory needs. • Professor Siegfried Drewes (my co-supervisor) at the Chemistry Department of the KwaZulu-Natal University, Pietermaritzburg. Vielen Dank FOr Ihren Beistand in meinem Studium und fOr die Zeit die Sie aufgeopfert haben, mit mir an meinen Problemen zu arbeiten. Es war ein besonderes Vorrecht mit so einem erfahrenen Akademiker zu arbeiten. Or Orde Munro must be thanked for the X-Ray crystallography and also for sharing his knowledge on this subject. Thank you also to Mr Martin Watson and Mr Craig Grimmer for NMR spectra done in Pietermaritzburg. Special thanks to you, Craig, for your patients even when you had to spend an hour or more with me in search for more information. It has been wonderful to work in Pietermarizburg and efficient laboratory staff like Mr Raj Somaru made it so much easier to get the work done. 3 • Or Neil Crouch at the National Botanical Institute, Durban, who collected all the plant material for this project and supplied me with photographs of the different plants. Thank you, Neil, for always being very helpful in providing me with the information I needed. • Professor Hannes van Staden at the Research Centre for Plant Growth and Development, University of KwaZulu-Natal, Pietermaritzburg. Baie dankie, prof, vir die positiewe gesindheid wat jy deurentyd geopenbaar het en dat jy gesorg het dat ek alles tot my beskikking gehad het om die biologiese werk te voltooi. Jy was 'n groot steun en bron van insig! A special thank you also to Or Esam Elgorashi for working side by side with me in the laboratory and for sharing his knowledge with me - it has really been a privilege working with such a meticulous person like you, Esam. The staff and students at the Research Centre have also been very helpful and friendly - thank you especially to Or Wendy Burnett for proofreading my biological chapters. • Professor Sarel Malan at the Pharmaceutical Chemistry Department, Potchefstroom University. Dankie vir jou behulpsaamheid en die vriendelike wyse waarop ek by die universiteit ontvang is, Sare!. Dit was 'n voorreg om na soveel jare weer saam te werk! Professor Faans Steyn at the Statistical Consultation Services, Potchefstroom. Dankie, prof, vir die tyd aan my werk afgestaan. Ek het altyd welkom gevoel om nog 'n vraag te kom vra. 4 Or Louis Fourie at the Chemistry Department, Potchefstroom University. Dankie, Louis, vir die vinnige en effektiewe afhandeling van die Massa Spektra! •I would also like to express my gratitude for a bursary from the Foundation for Research and Development which provided financial assistance during this study. • Mrs Jayne Leggat, my employer at the Howick Village Pharmacy. Thank you, Jayne, for bearing with me and for giving me more leave than that due to me. I'll never forget the support given to me by you and Pipsey! • Dankie aan my vriende en familie vir julle ondersteuning. Vir al die kere wat julle kinders opgepas het en gebel het om te hoor hoe dit gaan. 'n Spesiale dankie aan my ouers wat nooit aan my getwyfel het nie en my geleer het om nooit moed op te gee nie. • 'n Baie spesiale dankie aan my man, Andre en twee seuns, Francois en Philip, vir jare se opoffering en liefde - sander julle sou ek nooit hierdie projek kon voltooi het nie! 5 List of Abbreviations AlA - anti-inflammatory activity ACD - Advanced Chemistry Development AM 1 - Austin Method 1 AMA - antimicrobial activity AMP - adenosine monophosphate ax - axial BC - bacteriostatic concentration br d - broad doublet br s - broad singlet CE - Coulombic interaction energy CoA - coenzyme A COSY - correlated nuclear magnetic resonance spectroscopy COX - cyclooxygenase 0- density d - doublet dd - doublet of doublets ddd - double doublets DEPT - distortionless enhancement by polarization transfer DMSO - dimethyl sulfoxide DNA - deoxyribonucleic acid DP - dipole moment EDTA- ethylenediaminetetra-acetate EF - heat of formation eq - equatorial ES - aqueous phase energy FDA - United States Food and Drug Administration FSA - Flora of southern Africa HMBC - heteronuclear multiple bond coherence HMQC - heteronuclear multiple quantum coherence 6 HSQC - heteronuclear single quantum coherence Hz-hertz ICso - concentration needed to inhibit 50% of the enzyme INT - p-iodonitrotetrazolium violet IR - infra red J - coupling constant m - multiplet mlz - mass-to-charge ratio MH Broth - Mueller-Hinton Broth mM - millimole MM+ - molecular mechanics force field MMFF94 - Merck Molecular Force Field MR - molar refractivity NIE - total nonbonded interaction energy NMR - nuclear magnetic resonance NOESY - nuclear Overhauser enhancement spectroscopy NSAID - nonsteroidal anti-inflammatory drugs ORTEP - Oak Ridge Thermal Ellipsoid Program P- parachor POl - polarizability ppm - parts per million q - quartet QSAR - quantitative structure-activity relationships RDA - retro-Diels-Alder RI - refractive index s - singlet SA - surface area SE - strain energy ST - surface tension SV - volume of a space-filling model t - triplet 7 TE - vibrational entropy TLC - thin layer chromatography UV - ultra-violet VE - Van der Waals interaction energy VEL - vibrational enthalpy 8 List of Figures 1.1. The three basic structural types of homoisoflavanones 1.2. The structure of scillascillin 1.3. Two parent hydrocarbons important for this study 1.4. The spirocyclic nortriterpene, eucosterol/ (23S)-17a,23-epoxy-3~,29­ dihydroxy-27-nor-lanost-8-ene-15,24-dione, isolated from different Eucomis species 1.5. The spirocyclic nortriterpene, natalensis A, isolated from Merwilla natalensis . 1.6. The cholestane glycosides, (22S),3J3, 11 ~,22~-trihydroxycholest-5-ene 16J3-0-a-L-rhamnopyranoside and its two acetyl derivatives isolated from 0. saundersiae 1.7. Acylated cholestane glycosides isolated from 0. saundersiae 1.8. Cholestane bisdesmosides from 0. thyrsoides 1.9. Structure of 5~-16J3, 23-epoxy-22J3-hydroxy-18-oxocholesta-24-ene 3~-O -a-L-rhamnopyranosyl-(1 ~2)-~-O-glucopyranosyl-(1~2)-~-O-glucopyra­ noside 1.10. The structure of galtonioside A, isolated from Galtonia candicans 1.11. Comparison of the structures of a bufadienolide and a cardenolide 1.12. The structures of physodine C (X) and physodine 0 (XI) which were the first examples of 14-deoxybufadienolides 1.13. Structure of urginin, isolated from U. altissima 2.1. The structure of compound 1, punctatin 2.2. The structure of compound 2, (Z)-eucomin 2.3. The structure of compound 3, (E)-eucomin 2.4. The structure of compound 4, 3,9-dihydropunctatin 2.5. The structure of compound 5, 5-methoxy-7-hydroxy-3-(4'­ hydroxybenzyl)-4-chromanone 2.6. The structure of compound 6, 7-0-methyleucomol 9 2.7. The structure of compound 7, 3f3,17a-dihydroxycholest-5-en- 22-one 16f3-0-a-L-arabinopyranoside. 2.8. The structure of compound 8, the acetylation product of compound 7. 2.9. The structure of compound 9, sitosterol 3-0-f3-D-glucopyranoside 2.10. The NOESY correlations giving evidence of a f3-D-glucopyranoside 2.11. The structure of compound 10, 25R,5f3-spirostane-1 f3,3a-diol. 2.12. NOESY correlations observed in ring F of compound 10 2.13. Labelled ORTEP diagram (30% probability ellipsoids) of compound 10 2.14. Asymmetric unit from the crystal of compound 10 (two independent molecules) 2.15. Unit cell contents 2.16. The X-ray structure of 25R,5a-spirostane-1f3,3f3-diol, isolated by Patil et al. 2.17. Comparison of the basic structures of 25R,5a-spirostane-1 f3,3f3-diol and compound 10. 2.18. Comparison of F rings of compound 10 and 25R,5a-spirostane- 1f3,3f3-diol 2.19. The structure of compound 11, 16f3-acetoxyberscillogenin 2.20. The structure of compound 12, Iydenburgenin 2.21. The structure of compound 13, the acetylation product of compound 12.